Blocking junctional adhesion molecule C promotes the recovery of cisplatin-induced acute kidney injury

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1 ORIGINAL ARTICLE Koren J Intern Med 217;32: Blocking junctionl dhesion molecule C promotes the recovery of cispltin-induced cute kidney injury Sun Chul Kim, Yoon Sook Ko, Hee Young Lee, Myung-Gyu Kim, Sng-Kyung Jo, nd Won-Yong Cho Division of Nephrology, Deprtment of Internl Medicine, Kore University Anm Hospitl, Seoul, Kore Received : Februry 25, 216 Revised : April 23, 216 Accepted : April 25, 216 Correspondence to Won-Yong Cho, M.D. Deprtment of Internl Medicine, Kore University Anm Hospitl, 73 Inchon-ro, Seongbuk-gu, Seoul 2841, Kore Tel: Fx: E-mil: wonyong@kore.c.kr Bckground/Aims: Recent findings hve demonstrted the occurrence of neutrophil trnsendothelil migrtion in the reverse direction (reverse TEM) nd tht endothelil junctionl dhesion molecule C (JAM-C) is negtive regultor of reverse TEM. In this study, we tested the effects of JAM-C blocking ntibody on the resolution of kidney injuries nd inflmmtion in mouse model of cispltin-induced cute kidney injury (AKI). Methods: Cispltin ws dministered vi intrperitonel injection. A JAM-C blocking ntibody or control immunoglobulin G ws dministered intrperitonel t 1.5 mg/kg, with the injection being delyed until dy 4 following cispltin dministrtion to restrict the effect of ntibodies on recovery. Results: After cispltin injection, serum cretinine nd histologic injuries peked on dy 4. Tretment with JAM-C blocking ntibody on dys 4 nd 5 promoted the functionl nd histologic recovery of cispltin-induced AKI on dys 5 nd 6. Fcilitting recovery with JAM-C blocking ntibody correlted with significntly incresed circulting intercellulr dhesion molecule 1 + Tmm-Horsfll protein + neutrophils nd significntly decresed renl neutrophil infiltrtion, indicting tht fcilitting reverse the TEM of neutrophils from the kidney to the peripherl circultion prtilly medited the resolution of inflmmtion nd recovery. Conclusions: These results demonstrted tht reverse TEM is involved in the resolution of neutrophilic inflmmtion in cispltin-induced AKI nd tht JAM-C is n importnt regultor of this process. Keywords: Junctionl dhesion molecule C; Trnsendothelil nd trnsepithelil migrtion; Cispltin; Acute kidney injury INTRODUCTION Cispltin is one of the most effective chemotherpeutic gents ginst solid tumors, but renl toxicity occurs in 25% to 3% of ptients receiving the drug. Cispltin-bsed nephrotoxicity develops in dose-dependent mnner nd prevents optiml cispltin dosing for tumor tretment [1,2]. Cispltin tretment cn led to tubulointerstitil inflmmtion, resulting in cute kidney injury (AKI), nd its long-term use my cuse chronic fibrosis [3]. Inflmmtion plys n importnt role in cispltin nephrotoxicity, nd mny studies hve been con- Copyright 217 The Koren Assocition of Internl Medicine This is n Open Access rticle distributed under the terms of the Cretive Commons Attribution Non-Commercil License ( by-nc/3./) which permits unrestricted noncommercil use, distribution, nd reproduction in ny medium, provided the originl work is properly cited. pissn eissn

2 The Koren Journl of Internl Medicine Vol. 32, No. 6, November 217 ducted in ttempt to elucidte the mechnism of this inflmmtory response [4,5]. As sustined immune response my result in severe tissue dmge, resolution of inflmmtion my be n essentil step in tissue regenertion nd repir. However, the mechnism of recovery following inflmmtion hs not been clerly identified. Inflmmtory responses re chrcterized by the infiltrtion of neutrophils, mcrophges, nd nturl killer T cells into tissues. Neutrophils in prticulr re known to penetrte into tissues in the erly phse of inflmmtion, cusing n innte immune response [6]. To migrte from the vsculr lumen into inflmed tissues, neutrophils must pss through endothelil blood vessel cells, process known s trnsendothelil migrtion (TEM). The junctionl dhesion molecule C (JAM-C) hs been reported to block the movement of neutrophils from inflmed tissue into the systemic circultion, process referred to s reverse TEM. The genetic deletion of JAM-C or the use of JAM-C-blocking ntibody cn promote reverse TEM [7]. In recent study, neutrophils tht underwent reverse TEM were reported to express intercellulr dhesion molecule 1 (ICAM-1) [8]. When using JAM-C-blocking ntibody in n ischemic/reperfusion injury model, n increse of ICAM-1 + neutrophils in the blood ws observed [7]. These results suggest tht neutrophils infiltrting into inflmed tissues cn be removed through reverse TEM; thereby, mitigting the inflmmtory response. However, no reports hve documented the effect of JAM-C-blocking ntibody on cispltin-induced AKI. We therefore exmined the effect of JAM-C-blocking ntibody on the removl of tissue neutrophils in the recovery phse of cispltin-induced AKI. We lso investigted whether these chnges could mitigte inflmmtory responses in the kidney nd led to functionl nd histologicl improvement. METHODS Animls nd drugs Mle C57BL/6 mice (7- to 8-week-old) were purchsed from Orient (Chrles River Kore, Seoul, Kore) nd llowed unrestricted ccess to food nd wter before experimenttion begn. Animl cre complied with the rules of the Animl Cre Committee of Kore University for the use of lbortory nimls in reserch. Cispltin (Sigm-Aldrich, St. Louis, MO, USA) ws diluted in norml sline to finl concentrtion of 2 mg/ml. Cispltin-treted mice were given single intrperitonel injection of cispltin (18 mg/kg), while control group received the sme volume of norml sline. To test the effect of JAM-C blocking ntibody in the recovery phse of cispltin-induced AKI, either monoclonl nti-mouse JAM-C ntibody (clone H33, Millipore, Billeric, MA, USA) or control immunoglobulin G (IgG) ws dministered vi intrperitonel injection t 1.5 mg/kg on dy 4 nd 5 following cispltin injection. Mice were scrificed on dys 4, 5, or 6 dys fter cispltin dministrtion. Their blood ws collected by intrcrdic puncture, nd both kidneys were processed for histologicl exmintion nd RNA isoltion. Biochemicl nlysis nd histologicl exmintion Blood ure nitrogen (BUN) nd cretinine levels were mesured using Hitchi 747 utomtic nlyzer (Blck Scientific Inc., Bohemi, NY, USA). Renl tissue smples were fixed in 4% buffered prformldehyde nd embedded in prffin. After deprffiniztion, 5-mm sections were processed nd stined with periodic cid-schiff. Tubulr dmge ws defined by the observtions of tubulr epithelil swelling, loss of brush border, vcuolr degenertion, necrotic tubules, cst formtion, nd desqumtion. Tubulr dmge ws estimted by exmining 8 to 1 high-power fields (HPFs; 2) per section nd using scoring system bsed on the percentge of dmged tubules per field (1, < 25%; 2, 25% to 5%; 3, 5% to 75%; 4, > 75%). The men scores were compred between the groups. For the immunohistochemicl detection of neutrophils, kidney tissues were stined with n nti-ly6g (lymphocyte ntigen 6 complex, locus G) ntibody (ebioscience, Sn Diego, CA, USA). Eight to 1 HPFs ( 2) of the outer stripe of the outer medull were cptured, nd the men number of Ly6G-positive cells per HPF ws lso compred. Rel-time polymerse chin rection RNA ws extrcted from kidney tissues using TRIzol (Invitrogen, Life Technologies, Seoul, Kore) ccording to the mnufcturer s instructions. RNA ws purified using the RNesy Mini Kit (Qigen, Vlenci, CA,

3 Kim SC, et l. Effect of blocking JAM-C on cispltin nephrotoxicity USA). Then, 1 μg totl RNA ws reverse trnscribed in rection volume of 5 μl contining 1 RT buffer, 5.5 mm MgCl 2, 5 μm of ech deoxynucleotide, 2.5 μm rndom hexmers,.4 U/μL RNse inhibitor, nd U/μL MultiScribe Reverse Trnscriptse (TqMn Reverse Trnscription Regents, Applied Biosystems Inc., Foster City, CA, USA) t 25 C for 1 minutes, 48 C for 3 minutes, nd 95 C for 5 minutes. Subsequently, rel-time polymerse chin rection (PCR) ws performed with the icycler IQ Rel-time PCR Detection system (Bio-Rd, Hercules, CA, USA) under the mplifiction conditions of 4 cycles of 95 C, 15 seconds nd 6 C, 1 minute. We used 18S ribosoml RNA (TqMn ribosoml control regent, Applied Biosystems Inc.) s n internl control to normlize the dt. Flow cytometry To detect ICAM-1 + neutrophils in the blood, peripherl blood cells were stined with ntibodies ginst mouse grnulocyte receptor 1 (Gr-1) nd mouse ICAM-1 (ebioscience), or mouse ICAM-1 nd mouse Tmm-Horsfll protein (BIOSS ntibodies, Woburn, MA, USA). Four-color fluorescence nlyses were performed (FACSClibur, BD Bioscience, Sn Jose, CA, USA) to determine the percentges of ICAM-1 + reverse-trnsmigrted neutrophils nd ICAM-1 + Tmm-Horsfll protein + neutrophils in the peripherl blood. These dt were nlyzed using FlowJo softwre (Tree Str Inc., Ashlnd, CA, USA). Sttisticl nlysis The dt were presented s the men ± SE nd nlyzed using the Kruskl-Wllis test. A p <.5 ws considered sttisticlly significnt. RESULTS Effects of in vivo blockde of JAM-C on circulting ICAM-1 + neutrophils Neutrophils tht hve remigrted from inflmed tissue to the systemic circultion through the endothelil brrier re known to express ICAM-1. Our flow cytometry results showed tht, t 5 dys fter cispltin dministrtion, the number of ICAM-1 +, Gr-1 + neutrophils in the peripherl blood hd significntly incresed in the JAM-C blocking ntibody-treted group, compred with tht of the IgG-treted control group (Fig. 1). Shm Cis dy 4 Cis dy 5 Cis + JAM-C Ab dy 5 ICAM-1 Gr-1 No. of ICAM + neutrophils/1 5 cells 3, 2,5 2, 1,5 1, 5 Shm Cis dy 4 Cis dy 5 Cis + JAM-C Ab dy 5 Figure 1. Effect of n in vivo junctionl dhesion molecule C (JAM-C) blockde on circulting intercellulr dhesion molecule 1 (ICAM-1) + neutrophils. Flow cytometry results showed the number of ICAM-1 + grnulocyte receptor 1 + (Gr- 1 + ) reverse-trnsmigrted neutrophils in the peripherl blood significntly incresed in the JAM-C blocking ntibody (Ab)-treted group, compred with tht observed in the immunoglobulin G (IgG)-treted control group (n = 6 nimls per group). Cis, cispltin. p <.5, compred with the control IgG group

4 The Koren Journl of Internl Medicine Vol. 32, No. 6, November 217 Effects of in vivo blockde of JAM-C on circulting ICAM-1 +, Tmm-Horsfll protein + neutrophils To confirm the origin of the ICAM-1 + reverse-trnsmigrted neutrophils, we exmined their expression of the Tmm-Horsfll protein by flow cytometry. At 5 dys fter cispltin injection, more ICAM-1 +, Tmm-Horsfll protein + neutrophils were present in the JAM-C blocking ntibody-treted group thn in the control IgG group (Fig. 2). These results suggested tht the considerble increse in ICAM-1 + neutrophils in the peripherl blood ws due to their return to the systemic circultion from previously infiltrted kidney tissue. In vivo blockde of JAM-C resulted in fster resolution of neutrophilic inflmmtion To determine whether the increse in reverse-trnsmigrted neutrophils (ICAM-1 +, Gr-1 + neutrophils) in the peripherl blood led to decrese in kidney-infiltrted neutrophils, we lso estimted kidney-infiltrted neutrophils t 5 dys fter cispltin injection. Ly6G stining reveled tht, t 5 dys fter cispltin injection, the number of kidney-infiltrted neutrophils significntly decresed in the JAM-C blocking ntibody-treted group, compred with tht determined for the control IgG-treted group (Fig. 3A). We investigted the effects of the JAM-C blocking ntibody on the expression of kidney cytokines (interleukin 6 [IL-6] nd tumor necrosis fctor α [TNF-α]), using rel-time PCR. By dy 5, expression of the proinflmmtory cytokine IL-6 decresed significntly in the group treted with JAM-C blocking ntibody (Fig. 3B). TNF-α levels showed no sttisticlly significnt difference between groups. In vivo blockde of JAM-C resulted in fster functionl nd histologicl recovery BUN nd cretinine levels peked on the 4th dy of cispltin dministrtion nd subsequently decresed. On the 5th nd 6th dys, declines in the BUN nd cretinine levels were significntly higher in the JAM-C blocking ntibody-treted group, compred with the corresponding levels mesured in the control IgG-treted group (Fig. 4A). In ddition, the tubulr injury score mesured on the 5th dy of cispltin dministrtion ws lso less in the JAM-C blocking ntibody-treted group thn in the control IgG-treted group (Fig. 4B). Shm Cis dy 5 Cis + JAM-C Ab dy 5 Tmm-Horsfll protein 1, ICAM-1 No. of THP + ICAM + neutrophils/1 5 cells Shm Cis dy 5 Cis + JAM-C Ab dy 5 Figure 2. Effects of n in vivo junctionl dhesion molecule C (JAM-C) blockde on circulting intercellulr dhesion molecule 1 + (ICAM-1 + ), Tmm-Horsfll protein + (THP + ) neutrophils. After gting neutrophils bsed on their forwrd sctter nd side sctter properties, we prepred bivrite histogrm showing ICAM-1 + nd THP + cells. ICAM-1 +, THP + neutrophil levels incresed in the JAM-C blocking ntibody (Ab)-treted group, compred to those found in control immunoglobulin G (IgG)-treted group on dy 5 fter cispltin injection (n = 6 nimls per group). Cis, cispltin. p <.5, compred with control IgG group

5 Kim SC, et l. Effect of blocking JAM-C on cispltin nephrotoxicity A Men no. of Ly6G+ cell/hpf Shm Cis dy 5 Cis + JAM-C Ab dy 5 Fold difference compred to shm B IL-6 Cis dy 4 Cis dy 5 Cis + JAM-C Ab dy 5 TNF-α Figure 3. In vivo blockde of junctionl dhesion molecule C (JAM-C) resulted in fster resolution of neutrophilic inflmmtion. (A) The number of infiltrted neutrophils in the kidney significntly decresed in the JAM-C blocking ntibody (Ab)-treted group, compred to tht found in the control immunoglobulin G (IgG)-treted group on dy 5 fter cispltin injection (lymphocyte ntigen 6 complex, locus G [Ly6G] stin, 2; n = 6 nimls per group). (B) The expression of interleukin 6 (IL-6) significntly decresed in the JAM-C blocking Ab-treted group (n = 6 nimls per group). Tumor necrosis fctor α (TNF-α) levels showed no sttisticlly significnt differences between groups. Cis, cispltin; HPF, high-power field. p <.5, compred with the shm or control IgG group. DISCUSSION The mechnisms of cispltin-induced nephrotoxicity include DNA dmge, ltertion of the cellulr trnsport system, mitochondril dysfunction, oxidtive stress, inflmmtory responses, ctivtion of mitogen-ctivted protein kinses, ctivtion of poptotic pthwys, nd endothelil dysfunction [3]. Inflmmtory responses re prticulrly well known to medite cispltin-induced nephrotoxicity. Incresed inflmmtory cells nd proinflmmtory cytokines were observed in cispltin-induced AKI in previous studies [5,9,1]. In this study, increses in infiltrted neutrophils nd IL-6 levels were observed in kidney tissue t 4 dys fter cispltin dministrtion. These findings suggested tht immune cells nd inflmmtory responses were involved in cispltin-induced AKI. In ddition, previous dt showed tht infiltrted inflmmtory cells from the blood could mplify inflmmtory cytokine production nd chemokine secretion; thereby, promoting tissue dmge nd ultimtely reduction in renl function nd fibrosis [11]. For the induction of inflmmtion, neutrophils in the blood must infiltrte inflmed tissue by crossing the vsculr endothelil brrier. This process involves vriety of fctors, such s PECAM-1 (pltelet endothelil cell dhesion molecule 1), ICAM-2, CD99, ESAM (endothelil cell-selective dhesion molecule), nd junctionl dhesion molecules. To end inflmmtion, infiltrted inflmmtory cells such s neutrophils, mcrophges, nd nturl killer T-cells must be removed. Until recently, the resolution of inflmmtion ws thought to be pssive process relted to the disppernce of proinflmmtory stimuli. However, recent findings hve demonstrted the involvement of n ctive, tightly regulted process tht induces the reduction of proinflmmtory meditors nd the removl of inflmmtory cells [12,13]. Although the mechnisms of infiltrted neutrophil removl re known to include poptosis nd phgocytosis, recent dt hve shown tht infiltrted neutrophils cn be removed through their reentry into systemic circultion by reverse TEM [7,14,15]. JAM-C is locted in the endothelil cell junction nd regultes the unidirectionl TEM of neutrophils from the blood to tissues [16,17]. Woodfin et l. [7] presented rel-time confocl imges showing tht neutrophil TEM ws delyed or even reversed following ischemic/

6 The Koren Journl of Internl Medicine Vol. 32, No. 6, November Tubulr injury score A Shm Cis dy 5 Cis + JAM-C Ab dy Cis Cis + JAM-C Ab 12 BUN (mg/dl) Cretinine (mg/dl) B Shm Dy 4 Dy 5 Dy 6 Shm Dy 4 Dy 5 Dy 6 Figure 4. In vivo blockde of junctionl dhesion molecule C (JAM-C) resulted in fster functionl nd histologic recovery. (A) Declines in blood ure nitrogen (BUN) nd cretinine levels significntly incresed in the JAM-C blocking ntibody (Ab)-treted group, compred to those mesured in the control immunoglobulin G (IgG)-treted group (n =18 nimls per group). (B) Tubulr injury scores mesured on the 5th dy of cispltin dministrtion decresed in the JAM-C blocking Ab-treted group (lymphocyte ntigen 6 complex, locus G [Ly6G] stin, 2; n = 6 nimls per group). Cis, cispltin. p <.5, compred with the shm or control IgG group

7 Kim SC, et l. Effect of blocking JAM-C on cispltin nephrotoxicity reperfusion injury. These phenomen re relted to the disppernce of JAM-C t the endothelil cell junction. The genetic deletion of JAM-C or employing JAM-Cblocking ntibody promoted the reverse TEM of neutrophils [7]. Becuse this study ws imed t investigting the effects of JAM-C-blocking ntibody on the recovery phse of cispltin-induced AKI, the JAM-C-blocking ntibody ws dministered beginning t dy 4 postcispltin dministrtion, when mximum renl injury is normlly observed. On dy 5, the number of infiltrted neutrophils in the kidneys of nimls treted with JAM- C-blocking ntibody hd decresed. TUNEL (terminl deoxynucleotidyl trnsferse dutp nick end lbeling) stining ws performed to detect poptotic neutrophils in the kidney. No significnt differences were observed in the number of poptotic neutrophils detected between the JAM-C-blocking ntibody-treted group nd the control IgG-treted group (dt not shown). In ddition, on dy 5, in contrst to tissue neutrophils, ICAM- 1 + reverse-trnsmigrted neutrophils in the peripherl blood incresed in the JAM-C-blocking ntibody-treted group. To detect the origin of these ICAM-1 + neutrophils in the blood, we stined for the Tmm-Horsfll protein, which is relesed from the picl domin of the thick scending limb of the loop of Henle into the interstitium nd circultion during the recovery phse of kidney injury [18]. In this study, we observed tht ICAM- 1 +, Tmm-Horsfll protein + neutrophils incresed in the blood of the JAM-C-blocking ntibody-treted group. These results suggested tht blocking JAM-C fcilittes the reverse TEM of neutrophils in inflmed kidney tissue. IL-6, proinflmmtory cytokine, is known to increse in cispltin-induced AKI [9,1]. In this study, IL-6 levels were lso incresed on dy 4 fter cispltin injection, fter which tendency towrds decresed production ws observed. Significnt declines in IL-6 production were observed in JAM-C-blocking ntibody-treted nimls, compred to the control IgG-treted group on dy 5. This finding suggested tht the removl of tissue neutrophils by reverse TEM might fcilitte the reduction of inflmmtion. TNF-α lso plys centrl role in mounting inflmmtory responses by stimulting the expression of other inflmmtory meditors, such s trnsforming growth fctor β, RANTES (regulted on ctivtion, norml T cell expressed nd secreted), MIP-2 (mcrophge inflmmtory protein 2), monocyte chemottrctnt protein 1 (MCP-1), IL-1b, nd ICAM-1 during cispltin-induced nephrotoxicity [19]. However, in this study we did not observe sttisticlly significnt difference in TNF-α levels between the JAM-C-blocking ntibody-treted group nd the control IgG-treted group. This outcome my hve occurred becuse the TNF-α level ws lredy reduced on dy 5, regrdless of JAM-C ntibody tretment. Finlly, we observed tht declines in BUN nd cretinine levels were significntly enhnced in the JAM-Cblocking ntibody-treted nimls during the recovery phse of cispltin-induced AKI. In histologic exmintions, tubulr injury scores mesured on the 5th dy of cispltin dministrtion were lso less in the JAM-Cblocking ntibody-treted group. These results showed tht the removl of infiltrted tissue neutrophils nd the reduction of neutrophilic inflmmtion through the promotion of reverse TEM fcilitted functionl nd histologicl kidney recovery. During this process, JAM-C is n importnt regultor of reverse TEM in cispltin-induced AKI. However, it is possible tht the reduction of locl inflmmtory responses through the promotion of reverse TEM is ssocited with systemic inflmmtion. ICAM-1 + reverse-trnsmigrted neutrophils hve greter bility to generte rective oxygen species [8]. Woodfin et l. [7] demonstrted tht the number of ICAM-1 + neutrophils in the lung vsculture ws significntly higher in the JAM-C-blocking ntibody-treted group thn the control IgG-treted group in cremster muscle ischemic/reperfusion injury model. A more recent study showed evidence tht severe lung injury ws induced in JAM-C knock out pncretitis model mice [2]. In this study, we use JAM-C-blocking ntibody insted of JAM-C knockout mice. The use of low-dose JAM-C-blocking ntibody or injection of JAM-C during the recovery phse my fcilitte recovery from kidney injury, without significnt dmge to other orgns. Thus, further evlution of the dose or time dependence of JAM-C blocking my be required. In conclusion, the use of JAM-C-blocking ntibody during cispltin-induced AKI fcilitted reverse TEM. This reduced the number of infiltrted tissue neutrophils nd the inflmmtory response, resulting in the promotion of functionl nd histologicl kidney recov

8 The Koren Journl of Internl Medicine Vol. 32, No. 6, November 217 ery. These findings re expected to be useful for estblishing new therpeutic strtegies tht my ssist in the recovery from cispltin-induced AKI. KEY MESSAGE 1. Junctionl dhesion molecule C (JAM-C) is n importnt regultor of reverse trnsendothelil migrtion (TEM) in cispltin-induced cute kidney injury. 2. In vivo blockde of JAM-C ws ssocited with significntly incresed circulting intercellulr dhesion molecule 1 + Tmm-Horsfll protein + neutrophils nd significntly decresed renl neutrophil infiltrtion. 3. The removl of infiltrting tissue neutrophils through the promotion of reverse TEM fcilitted functionl nd histologicl kidney recovery. Conflict of interest No potentil conflict of interest relevnt to this rticle ws reported. REFERENCES 1. dos Sntos NA, Crvlho Rodrigues MA, Mrtins NM, dos Sntos AC. Cispltin-induced nephrotoxicity nd trgets of nephroprotection: n updte. Arch Toxicol 212;86: Peres LA, d Cunh AD Jr. Acute nephrotoxicity of cispltin: moleculr mechnisms. J Brs Nefrol 213;35: Snchez-Gonzlez PD, Lopez-Hernndez FJ, Lopez-Novo JM, Morles AI. An integrtive view of the pthophysiologicl events leding to cispltin nephrotoxicity. Crit Rev Toxicol 211;41: Choi DE, Jeong JY, Lim BJ, Lee KW, Shin YT, N KR. Pretretment with drbepoetin ttenutes renl injury in rt model of cispltin-induced nephrotoxicity. Koren J Intern Med 29;24: Kng KP, Kim DH, Jung YJ, et l. Alph-lipoic cid ttenutes cispltin-induced cute kidney injury in mice by suppressing renl inflmmtion. Nephrol Dil Trnsplnt 29;24: Kelly KJ, Willims WW Jr, Colvin RB, et l. Intercellulr dhesion molecule-1-deficient mice re protected ginst ischemic renl injury. J Clin Invest 1996;97: Woodfin A, Voisin MB, Beyru M, et l. The junctionl dhesion molecule JAM-C regultes polrized trnsendothelil migrtion of neutrophils in vivo. Nt Immunol 211;12: Buckley CD, Ross EA, McGettrick HM, et l. Identifiction of phenotypiclly nd functionlly distinct popultion of long-lived neutrophils in model of reverse endothelil migrtion. J Leukoc Biol 26;79: Fubel S, Lewis EC, Reznikov L, et l. Cispltin-induced cute renl filure is ssocited with n increse in the cytokines interleukin (IL)-1bet, IL-18, IL-6, nd neutrophil infiltrtion in the kidney. J Phrmcol Exp Ther 27;322: Kim MG, Yng HN, Kim HW, Jo SK, Cho WY, Kim HK. IL-1 medites rosiglitzone-induced kidney protection in cispltin nephrotoxicity. J Koren Med Sci 21;25: Rmesh G, Reeves WB. TNFR2-medited poptosis nd necrosis in cispltin-induced cute renl filure. Am J Physiol Renl Physiol 23;285:F61-F Henson PM. Dmpening inflmmtion. Nt Immunol 25;6: Serhn CN. Resolution phse of inflmmtion: novel endogenous nti-inflmmtory nd proresolving lipid meditors nd pthwys. Annu Rev Immunol 27;25: Sverymuttu SH, Peters AM, Keshvrzin A, Revy HJ, Lvender JP. The kinetics of 111indium distribution following injection of 111indium lbelled utologous grnulocytes in mn. Br J Hemtol 1985;61: Wei Q, Dong G, Chen JK, Rmesh G, Dong Z. Bx nd Bk hve criticl roles in ischemic cute kidney injury in globl nd proximl tubule-specific knockout mouse models. Kidney Int 213;84: Aurrnd-Lions M, Lmgn C, Dngerfield JP, et l. Junctionl dhesion molecule-c regultes the erly influx of leukocytes into tissues during inflmmtion. J Immunol 25;174: Brdfield PF, Scheiermnn C, Nourshrgh S, et l. JAM-C regultes unidirectionl monocyte trnsendothelil migrtion in inflmmtion. Blood 27;11: El-Achkr TM, McCrcken R, Liu Y, et l. Tmm-Horsfll protein trnsloctes to the bsolterl domin of thick scending limbs, interstitium, nd circultion during

9 Kim SC, et l. Effect of blocking JAM-C on cispltin nephrotoxicity recovery from cute kidney injury. Am J Physiol Renl Physiol 213;34:F166-F Rmesh G, Reeves WB. TNF-lph medites chemokine nd cytokine expression nd renl injury in cispltin nephrotoxicity. J Clin Invest 22;11: Wu D, Zeng Y, Fn Y, et l. Reverse-migrted neutrophils regulted by JAM-C re involved in cute pncretitis-ssocited lung injury. Sci Rep 216;6: